The present invention provides a process for removing glucoamylase from beer. The term "beer" as used in the present specification and claims is a brewed fermentation product produced from malted cereal grains (usually the chief cereal grain is barley) and hops as the main starting materials and includes many types of brewed beverages. Such beverages include, but are not limited to, lager, pilsner, Dortmund and Munich beers, as well as top fermented beverages such as ale, porter, and stout.
Although the details may vary somewhat from brewery to brewery, a generally representative procedure for making beer is as follows: (1) Ground malt (grist) is placed in a mash tun for saccharification together with water, and while the temperature is gradually raised from about 45.degree.-55.degree. C. to about 75.degree.-80.degree. C. over a period of from about 2 to about 3 hours, starch in the malt is decomposed into sugars such as maltose, dextrin, and the like; (2) The resulting mash after saccharification is filtered to obtain a clear malt liquor (wort); (3) Hops are added to the filtered malt liquor and boiled for about an hour or two; (4) The hot wort is transferred to a precipitation tank and, after removing hot coagulates, cooled to 5.degree.-10.degree. C.; (5) Yeast is added to the cooled wort, and oxygen is supplied to promote the growth of the yeast; (6) Fermentation is effected at temperatures of about 10.degree. C. for about a week (primary fermentation); (7) Secondary fermentation and aging of the resulting beer are effected in a storage tank at low temperatures of about 0.degree. C. for one to two months; (8) The beer is then filtered and packed into containers (usually bottles, cans, or barrels).
A typical lager sweet wort comprises a complex mixture of starch derived carbohydrates, which are classified as fermentable or nonfermentable according to whether they can be converted into ethanol by brewer's yeast. The fermentable carbohydrates are formed by hydrolysis of grain starches by two enzymes, .alpha.- and .beta.- amylase, derived from malted barley. In most American lagers malted barley also serves as the predominant starch source while a smaller proportion is derived from nondiastatic adjunct grains. In the United States, corn and #4 brewer's rice are the predominant adjuncts.
All grain starches are glucose homopolymers in which the glucose residues are linked by either .alpha.-1,4 or .alpha.-1,6 bonds. During the mashing cycle the starches are first solubilized and then a portion of the solubilized large starch molecules are predominantly hydrolyzed to three low molecular weight sugars which brewer's yeast can ferment to ethyl alcohol. The major fermentable sugars are glucose, maltose, and maltotriose while traces of sucrose and fructose are also present. The nonfermentable or limit dextrin fraction consists of all sugars of a higher degree of polymerization (DP) than maltotriose. The bulk of the limit dextrin fraction is composed of polysaccharides which are greater than 10,000 molecular weight.
As indicated above, the hydrolysis of the grain starches is catalyzed by two amylases endogenous to malted barley. One, .alpha.-amylase, is an endoamylase which randomly cleaves .alpha.-1,4 bonds in the interior of the raw, largely insoluble starch molecules, fragmenting them into large but soluble polysaccharides termed dextrins. The second, .beta.-amylase, is an exoamylase which sequentially cleaves .alpha.-1,4 bonds from the nonreducing end of these dextrins producing the three fermentable sugars described above. Both enzymes are inactive towards the .alpha.-1,6 linkages (branch points) of the starches (i.e., they are unsuitable to debranch the starch molecule) and this results in the formation of the limit dextrins described above.
After completing the mash cycle, the spent grains are removed by passing the mash through a lauter tun to obtain the clarified lager sweet wort. The wort is then transferred to a brew kettle and boiled vigorously for about 1 to 2 hours to inactivate the malt enzymes. It is then cooled, pitched with yeast, and fermented at temperatures ranging from about 8.degree. C. to about 16.degree. C. to convert the three sugars described above to ethanol. The composition of the wort can vary depending on the starting materials, mash cycle, and other variables. The carbohydrate composition of a typical wort comprises from about 65 to about 80 percent fermentable sugars, and from about 20 to about 35 percent limit dextrins. During fermentation much of the fermentable fraction is converted to ethanol. The final ethanol concentration at the end of fermentation ordinarily ranges from about 3 to about 6 percent w/w. The limit dextrins are not converted during fermentation and form the bulk of the dissolved solids, commonly referred to as real extract, in the final beer.
Reduced calorie beers have recently become popular in the United States and elsewhere. These beers may be formulated by (1) reducing both the alcohol and real extract concentrations in the beer to attain the desired calorie level, or (2) by hydrolyzing the limit dextrins with exogenous enzymes, one component of which is capable of debranching the limit dextrins. The latter method is advantageous since it allows one to attain the desired calorie level with a minimum reduction of the alcohol content of the packaged product. The enzyme most commonly used to hydrolyze the limit dextrins is glucoamylase, also known as amyloglucosidase or exo-1,4,.alpha.-glucosidase, which is a nonspecific exoamylase derived from a variety of fungal sources (e.g., Aspergillus niger, Rhizopus delemar, etc.) Glucoamylase is active versus both .alpha.-1,4 and .alpha.-1,6 linkages and therefore is capable of completely hydrolyzing starch to glucose. It attacks the starch molecule from the nonreducing end producing glucose as substantially the sole end product. It is also active versus starch derived oligosaccharides, e.g., maltose, maltotriose, isomaltose, etc. Most commercial glucoamylase is isolated from the mold Aspergillus niger. The glucoamylase produced by this organism is extracellular and is a glycoprotein containing approximately 16 percent carbohydrate.
In theory, glucoamylase may be added at any time during the brewing process. In practice however, brewers usually prefer to add glucoamylase during fermentation because the fermentation process itself requires from about 6 to about 15 days, depending on pitching rate, fermentation temperature, etc. The brewhouse operations, in contrast, are of much shorter duration (about 2 to 4 hours per brew) and operate under tight scheduling constraints. Therefore debranching enzymes are usually employed as fermentation adjuncts as disclosed in U.S. Pat. No. 3,379,534, and the limit dextrins are hydrolyzed to fermentable sugars, which the yeast converts to ethanol. These beers ferment to a lower specific gravity due to increased alcohol and decreased real extract than would the same beer without exogenous enzymes. Such beers are referred to as super attenuated beers.
A fermenting beer stream during primary fermentation contains a large concentration of suspended solids which originate from several sources. When the wort is cooled after kettle boil, a heavy precipitate forms which is allowed to settle out in a tank. The precipitate (a mixture of protein, carbohydrate, etc.) is referred to as traub and the settling process is referred to as hot-break. Traub separation is not complete during the hot-break, and its formation continues even during fermentation. Additionally, the wort or beer is pitched with a large (on the order of 1.times.10.sup.7 cells per milliliter of wort) concentration of brewer's yeast at the beginning of the fermentation. The yeast typically multiplies to six to nine times its original concentration at high kraeusen and then settles out as the specific gravity decreases toward the end of fermentation.
It is economically necessary for brewers to reclaim most of the expanded yeast crop at the end of fermentation to repitch fresh wort in order to supplement the crop produced by primary propagation. Ordinarily brewers are able to pitch about 3 to 6 fresh fermentations with the yeast reclaimed from one fermenter.
In addition to traub formation and yeast multiplication, there are two other major changes that occur during fermentation: (1) large quantities of carbon dioxide are evolved during active fermentation, and (2) the specific gravity of the beer decreases markedly.
The glucoamylase may be immobilized on a water insoluble solid support and brought into contact with the wort or beer. Several types of immobilized enzyme reactors have been described in the literature. The most prevalent are those in which the enzymes have been immobilized on particulate carriers. These include: (1) batch stir systems in which the immobilized adduct is stirred in the wort or beer and is later recovered by filtration, (2) plug flow reactors, also known as fixed bed reactors, in which the immobilized adduct is packed in a column and the wort or beer is passed through it in a manner similar to a column chromatography operation, (3) fluidized bed reactors, which are similar to plug flow reactors except that the wort or beer is circulated into the bottom of the column at sufficiently greater flow rate to fluidize the bed.
Reactors based on glucoamylase immobilized on particulate carriers are not suitable for processing a beer during primary fermentation for several reasons:
(1). The batch stir system is impractical since it would require that the yeast and the immobilized derivative be separated from each other at the end of fermentation in order to recover and reuse both the enzyme conjugate and the yeast cream. This separation would prove both difficult and costly.
(2). Plug flow reactors do not support flow of wort or beer streams containing large and variable amounts of suspended solids typical of a fermenting lager stream. Such reactors plug rapidly as the solids accumulate at the entrance face of the immobilized glucoamylase bed.
(3). Fluidized beds are impractical since the density of the fermenting beer stream is continuously decreasing during fermentation. Inasmuch as particle fluidization is dependent on the density of the wort or beer, the flow rate would have to be continuously increased throughout fermentation to compensate for the density decrease in order to keep the bed fluidized. In addition, during active fermentation the large quantities of carbon dioxide evolved would disrupt the even flow of liquid and make fluidization more difficult and possibly would channel the bed. Most fluidized bed reactors contain support retainers at both ends in order to prevent flow of the immobilized glucoamylase back into the wort or beer feed tank or into the product receiver. The suspended solid could accumulate at the retainers and block flow. Finally, at the end of fermentation the immobilized glucoamylase would have to be separated from the yeast that would be entrained with the residual beer.
In a effort to overcome the difficulties inherent in particulate systems, other types of reactors have been developed in which the glucoamylase has been immobilized by attachment to monolithic solid supports. The construction, configuration, and arrangement of such monolithic reactors vary from one system to another, but in general the wort or beer is brought into contact with the glucoamylase so immobilized.
One class of monolithic reactor is based on the immobilization of glucoamylase by attachment to various membranes. These membrane reactors suffer from the lack of strength of the membranes employed and the required backing on large amounts of the membrane support materials.
Another class of monolithic reactors is based on the immobilization of glucoamylase by attachment to ceramic monoliths having a lattice of generally parallel channels. The channels have cross-sectional linear dimensions on the order of from about 50 to about 3000 micrometers so that the flow of yeast and other undissolved solids present in the fermenting stream will pass through without being obstructed and so that there is sufficient surface area within a reactor of reasonable size to attach a reasonable amount of glucoamylase. Laboratory-sized reactors of this type function well, but the ceramic supports are very expensive and they are fragile. The capital expenditures for production-sized reactors are believed to be enormous.
By far the simplest manner of using glucoamylase is to dissolve the requisite amount in the wort or beer to be treated. This method has been widely employed for pasteurized beers since the heat of pasteurization destroys the glucoamylase. Glucoamylase has not been used in the production of draught beers because the production of these beers does not employ a pasteurization step; hence there is no heat available to destroy the glucoamylase.